This invention occurs with the need of more effective ways for the treatment of chronic wounds, especially in diabetic patients.
It is estimated that 15% of the diabetic patients have nonhealing foot ulcerations. In recent years, there have been efforts to develop new advanced methodologies to heal chronic wounds, including the use of topic growth factors or cell-based therapies. In case of stem cells, the treatment is based in the assumption that topical application of stem cells modulates the healing response. Recent data show that healthy adult stem/progenitor cells improve the healing of diabetic chronic wounds. It has been shown that peripheral blood-derived CD34+ cells, but not CD34− cells, can accelerate the vascularization and healing of diabetic wounds (Schatteman G C et al, 2003). However, the vasculogenic potential of adult blood-derived cells appears to be reduced by diabetes. Recent studies tried to overcome this issue by using fetal (Invernici G et al., 2009) or adult mesenchymal stem cells (Tredget E E et al., 2007), yet, the isolation of stem cells from fetal aorta poses several problems for future clinical application, while mesenchymal stem cells isolated from diabetic patients might have impaired properties due to ageing and disease. In case of fetal aorta stem cells, the results indicate that the stem cells released factors (e.g. VEGF and IL-8), which stimulate angiogenesis and activate Wnt signaling, promoting the healing of diabetic ischemic ulcers.
Human umbilical cord blood (UCB) can be a potential source of healthy endothelial progenitor cells for the healing of chronic wounds in diabetic patients. These cells are obtained noninvasively, can be stored for more than 15 years without loosing biological properties, and they have low immunogenicity, which makes them an interesting candidate for allogeneic transplantation. Improvement in wound healing has been reported recently in two human non-diabetic patients who received topically UCB-derived CD34+ cells in a fibrin gel (Dejana A M et al., 2004). Despite this potential, human umbilical cord blood stem cells have not been used for wound healing in diabetic patients, whose healing process is impaired or even inexistent. In this case, the stem cells may need the stimulation of bioactive agents and cell-cell interactions for survival and therapeutic effect. This is the focus of the present invention.
In the present patent, the therapeutic effect of CD34+ cells or the combination of CD34+ cells with CD34+-derived ECs was tested in streptozotocin-induced diabetic mice, which have previously been used to study the effect of cell-based therapies on wound healing (Invernici G et al., 2009, Wu Y, 2008). Importantly, no study has documented the regenerative potential of UCB-derived CD34+ cells in the context of diabetic wounds. Here, we show that the transplantation of CD34+ cells together with CD34+-derived ECs, but not CD34+ cells alone, in fibrin gels enhances wound healing, compared to wounds covered with gel alone or PBS. A substantial fraction of cells (more than 90%) died over a 3 day period. This is in line with other studies showing a dramatic decline of stem cells at the injury site (Invernici G et al., 2009)]. Even so, a therapeutic benefit was observed over the 10 day period in our study, likely due to factors secreted by both cells, which increased neovascularization and decreased the inflammatory reaction. Our results show that cytokines IL-10 and IL-17 are only secreted when both cells are co-cultured. IL-10 is a potent immunosuppressant of monocyte/macrophage functions, inhibiting the production of pro-inflammatory cytokines. Our Multiplex results also indicate that the level of INF-α, a pro-inflammatory cytokine, was lower for wounds treated with CD34+ cells and ECs, or only CD34+ cells encapsulated in fibrin gel.
Regarding prior art, there are several patents in the field of Regenerative Medicine that use some of the components of the present invention, but provide methods and compositions that are substantially different. For example, the patent application entitled “Using of scaffold comprising fibrin for delivery of stem cells” (US2010/0028311A1) provides, in part, compositions and methods for treating ischemia in a subject in need thereof. The method consisted in the administration of a fibrin scaffold or fibrin clot comprising stem cells (CD34+ cells). However, the invention did not describe methods to improve the viability, vascular differentiation and therapeutic effect of the CD34+ cells.